bacteriophages φx174  (ATCC)

Journal: Biotechnology Journal

Article Title: Analytical Centrifugal Ultrafiltration as a Tool for High Throughput Process Development in Virus Removal Filtration

doi: 10.1002/biot.70227

Figure Lengend Snippet: A. MS2 phage clearance at varying rotor speeds; B. Images of filter surface after centrifugal ultrafiltration at 5000 rpm, including nanocellulose‐based filter paper (upper panel) and support paper (lower panel); C. ΦX174 phage clearance at varying rotor speeds; D. LRV MS2 phage clearance to half‐volume at varying rotor speeds. Dotted line represents 4 log 10 virus clearance threshold. Dotted line represents 4 log 10 virus clearance threshold.

Article Snippet: Bacteriophages ΦX174 (ATCC 13706‐B1) and MS2 (ATCC 15597‐B1), along with their respective host Escherichia coli (Migula) Castellani and Chalmers bacterial strains C (ATCC 13706) and C‐3000 (ATCC 15597), were procured from ATCC (Manassas, VA).

Techniques: Virus

Journal: STAR Protocols

Article Title: Protocol for detecting interactions between intrinsically disordered proteins and long DNA substrates by electrophoretic mobility shift assay

doi: 10.1016/j.xpro.2025.103968

Figure Lengend Snippet: An example outcome of an EMSA with 0–0.4 μM IDR protein and 0.2 nM ssDNA substrate (ΦX174) (A) Free DNA was separated from IDR-bound DNA on a 1% agarose gel. A prominent upwards band shift occurs when IDR-DNA complexes are formed. Bovine serum albumin (BSA) was combined with ssDNA substrates at the highest corresponding concentration to the IDR of interest (0.4 μM) as a negative control for DNA-binding. Lane numbers are specified at the bottom of the gel. (B) IDR-bound DNA in lanes 2–7 from panel (A) was quantified relative to the sample that contained no protein (lane 1) in the EMSA gel. Data was fit with a Hill-Langmuir equation and a dissociation constant (K D ) was obtained from this curve. K D describes the protein concentration at which 50% DNA is bound. Data represents the replicate shown in panel A.

Article Snippet: ΦX174 virion ssDNA (5386 nt) , New England Biolabs , Cat # N3023S.

Techniques: Agarose Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Concentration Assay, Negative Control, Binding Assay, Protein Concentration

Journal: bioRxiv

Article Title: Generative design of novel bacteriophages with genome language models

doi: 10.1101/2025.09.12.675911

Figure Lengend Snippet: (A) Phage genome design workflow. (B) Genetic architecture of our design template, ΦX174, a Microviridae phage that uses E. coli C as a host. (C) For our generation strategy, we specialized Evo 1 and Evo 2 on Microviridae genomes through supervised fine-tuning (SFT) to enhance its ability to generate ΦX174-like sequences. (D) Sequence logos showing conserved nucleotides at the start of ΦX174 variant genomes compared to Microviridae genomes in our training data. (E) Increasing the number of ΦX174 nucleotides in the prompt quickly improves recall with the SFT models whereas the base models fail to recall ΦX174 across all prompt lengths. (F) Design constraints selected for genome filtering, with thresholds (blue) chosen against natural Microviridae distributions (gray) and ΦX174 (dotted line). (G) Benchmarking of six gene prediction methods on the genome of ΦX174 shows that overlapping genes are systematically missed, requiring us to create a new method that predicts all genes in ΦX174. (H) Final filtering and evaluation steps for generated genomes. (I) Maximum sequence retention rate across Evo 1 and 2 SFT-generated sequences after applying design constraints, with natural Microviridae , scrambled Microviridae , and ΦX174 variant (vars.) controls. The selected design constraints retain ΦX174-like sequences across quality control, tropism, and diversification filters. (J–K) Generated sequences have high Shannon diversity after tropism filtering (J) and maintain a high retention rate even after further diversification filtering (K) . Diversity and retention rate both increase with generation temperature and prompt length. Microviridae , scrambled Microviridae , and ΦX174 variants are shown for comparison. 𝑛 = 1000 sequences per parameter combination.

Article Snippet: ΦX174 am3 cs70 RFI DNA (NEB #N3021) was PCR-amplified into separate sets of two or three fragments (File S1).

Techniques: Sequencing, Variant Assay, Generated, Control, Comparison

Journal: bioRxiv

Article Title: Generative design of novel bacteriophages with genome language models

doi: 10.1101/2025.09.12.675911

Figure Lengend Snippet: (A) Final generated phage candidates meet our quality criteria while capturing abundant sequence diversity. kb, kilobase; Med., Medium; Comp. Complete. (B) Many generated sequences encode genes with low sequence identity to ΦX174, resulting in breaks in gene synteny. (C) A heatmap of total gene count versus number of syntenic genes shows that most sequences contain a single-gene break in synteny to ΦX174, balancing conservation with novelty. (D) Workflow for experimental validation of generated phage genomes. (E) E. coli C transformed with synthesized ΦX174 genome assemblies show that phage plaques robustly form across assembly conditions. In contrast, the same genomes but with loss-of-function mutations in lysis genes do not form plaques. ng, nanogram; frag., fragment. (F) Growth curves of E. coli C transformed with no phage (gray), ΦX174 lysis mutant (mut., light blue), or wild-type ΦX174 (dark blue) genome assembly reveal strong growth inhibition by ΦX174. Data point, mean OD 600 value; error bar, standard deviation; 𝑛 = 3 growth replicates. (G) Growth curves of generated genome assemblies transformed in E. coli C exhibiting strong growth inhibition. (H) Representative titrations of propagated phage candidates. (I) Growth inhibition measured by OD 600 at 6 hours after infection of E. coli cultures with no phage, ΦX174, or generated phages shows that ΦX174 and generated phages inhibit growth in the target strain E. coli C, and in E. coli W, but not in six other strains tested, demonstrating the robustness of tropism filtering. Each column is an infection replicate.

Article Snippet: ΦX174 am3 cs70 RFI DNA (NEB #N3021) was PCR-amplified into separate sets of two or three fragments (File S1).

Techniques: Generated, Sequencing, Biomarker Discovery, Transformation Assay, Synthesized, Lysis, Mutagenesis, Inhibition, Standard Deviation, Infection

Journal: bioRxiv

Article Title: Generative design of novel bacteriophages with genome language models

doi: 10.1101/2025.09.12.675911

Figure Lengend Snippet: (A) Synteny plot of ΦX174 and functional generated bacteriophages, highlighting hundreds of synonymous (light blue), nonsynonymous (dark blue), and noncoding (red) mutations compared to ΦX174. Genomes are in order by name. (B) Average nucleotide (nt) mutational frequencies, normalized to length, of structural proteins, non-structural proteins, and regulatory elements across the generated phage genomes show that gene J and regulatory elements are mutational hotspots. (C) Generated genomes exhibit a range of lengths. Dotted line, genome length of ΦX174; white dot, median; gray box, interquartile range (IQR); whiskers, 1.5× IQR. (D) Percent sequence identity and number of novel mutations of functional (blue) and non-functional (gray) generated sequences compared to their top nucleotide BLAST hit in the Microviridae training data. (E) Neighbor-joining phylogenetic tree of functional generated phages (light blue) and representative Microviridae phages (dark blue and pink). (F) Percent cumulative sequence coverage of generated phages highlights that mutations in most generated phages cannot be completely attributed to mutations seen in nature. The sequences were aligned to sequences by nucleotide BLAST in the core_nt database until all nucleotides were accounted for or there were no significant hits for remaining nucleotides. (G–H) Synteny plot (G) and detailed view of gene J (H) and its surrounding intergenic regions of ΦX174, Evo-Φ36, and phage G4, with single nucleotide variations (SNVs) compared to ΦX174 highlighted in blue. (I) Cryo-EM density map of Evo-Φ36 virion, highlighting individual subunits of the spike (pink) and capsid (yellow). Remaining spikes (light blue) and capsids (gray) are shown. (J) Interior surface view of the capsid (F, gray) and spike (G, not visible) pentamers of Evo-Φ36 (left) and ΦX174 (right), with their cognate J proteins (purple) reveals distinct capsid interactions and putative genome packaging modes between the two phages as modeled into the cryo-EM density map. (K) Asymmetric units including F, G, and J of Evo-Φ36 (left) and ΦX174 (right) show resolved residues of J.

Article Snippet: ΦX174 am3 cs70 RFI DNA (NEB #N3021) was PCR-amplified into separate sets of two or three fragments (File S1).

Techniques: Functional Assay, Generated, Sequencing, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Generative design of novel bacteriophages with genome language models

doi: 10.1101/2025.09.12.675911

Figure Lengend Snippet: (A) Phage fitness competition assay workflow. (B–C) In three competitions, generated phages and ΦX174 competed head-to-head in E. coli C at equal multiplicity of infection (MOI). We tracked cumulative fold change (log 2 (FC)) of sequencing read counts over six hours (B) . Many generated phages matched or surpassed ΦX174’s performance at various time points (C) , indicating a higher relative fitness. Growth curves of infected E. coli C populations show corresponding suppression of bacterial growth. Rectangular boxes, enlarged plots in (C) ; arrowheads, sequencing sample extraction time points. Dotted line, cumulative log 2 (FC) of ΦX174. (D) Area under the curve (AUC) of the cumulative log 2 (FC) of phage read counts shows that generated phages outcompeted ΦX174 over the whole time course. Statistical significance was determined by one-way ANOVA with Tukey HSD (*𝑝-adj < 0.05). Bar height, mean; error bar, standard deviation; circles, 𝑛 = 3 competitions; dotted line, AUC of 0. (E) Growth dynamics of E. coli C infected with generated phages and ΦX174 individually show that several generated phages exhibit lower minimum population density after infection, steeper decline in host growth rate, and shorter time to minimum population density, together indicating stronger lytic capabilities. Statistical significance was determined by one-way ANOVA with Tukey HSD (*𝑝-adj < 0.05; **𝑝-adj < 0.01; ***𝑝-adj < 0.001). Bar height, mean; error bar, standard deviation; circles, 𝑛 = 3 infections; dotted line, mean value of ΦX174.

Article Snippet: ΦX174 am3 cs70 RFI DNA (NEB #N3021) was PCR-amplified into separate sets of two or three fragments (File S1).

Techniques: Competitive Binding Assay, Generated, Infection, Sequencing, Extraction, Standard Deviation

Journal: bioRxiv

Article Title: Generative design of novel bacteriophages with genome language models

doi: 10.1101/2025.09.12.675911

Figure Lengend Snippet: (A) Whole-genome sequencing of three ΦX174-resistant E. coli C strains revealed mutations in the waa operon absent in susceptible E. coli C, which functions in lipopolysaccharide synthesis. (B) Experimental setup for evolving phage counter-resistance by serially passaging cocktails of generated phages and ΦX174, or ΦX174 alone, on susceptible and resistant E. coli C. (C) Growth curves show that ΦX174 alone fails to overcome resistance, whereas generated phage cocktails suppress growth of all resistant cultures within five passages. Checkered flag, first passage with growth inhibition. (D–E) Alignments of generated phages against the predominant resistant phages Evo-ΦR1 (D) , Evo-ΦR2 (E) capable of infecting resistant strain 1 and 2, respectively, show that they are derived from generated genomes. Generated phages used in the alignments are those with the longest identical sequences (light blue) without single nucleotide variations (SNVs; dark blue) to each resistant phage such that they collectively minimize the number of novel mutations (yellow) observed in the resistant phage. Major capsid and spike proteins of each resistant phage aligned to ΦX174 major capsid and spike proteins are below, with synonymous (blue) and nonsynonymous mutations (pink) relative to ΦX174. (F) AlphaFold 3 (AF3) predictions of capsid (light blue) and spike (light gray) pentamers show that most capsid and spike mutations appear on the exterior of resistant phages. Nonsynonymous and novel nonsynonymous mutations relative to ΦX174 are highlighted in pink and yellow, respectively. pLDDT, predicted local distance difference test score; ipTM, interface predicted template modeling score; pTM, predicted template modeling score.

Article Snippet: ΦX174 am3 cs70 RFI DNA (NEB #N3021) was PCR-amplified into separate sets of two or three fragments (File S1).

Techniques: Sequencing, Passaging, Generated, Inhibition, Derivative Assay